Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition. Dihydrofolate reductase from with its two substrates, dihydrofolate (right) and NADPH (left), bound in the active site. ... HIV protease in a complex with the protease inhibitor ritonavir. ...

Enzyme units

Amounts of enzymes can either be expressed as molar amounts, as with any other chemical, or measured in terms of activity.

Enzyme activity = moles converted per unit time = rate × reaction volume. Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, which should be specified. The SI unit is the katal, 1 katal = 1 mol s^-1, but this is an excessively large unit. A more practical and commonly-used value is 1 enzyme unit (EU) = 1 μmol min^-1 (μ = micro, x 10^-6). 1 U corresponds to 16.67 nanokatals.

The specific activity of an enzyme is another common unit. This is the activity of an enzyme per milligram of total protein (expressed in μmol min^-1mg^-1). Specific activity gives a measurement of the purity of the enzyme.

Types of assay

All enzyme assays measure either the consumption of substrate or production of product over time. A large number of different methods of measuring the concentrations of substrates and products exist and many enzymes can be assayed in several different ways. Biochemists usually study enzyme-catalysed reactions using four types of experiments:^[1]

(1) Initial rate experiments. When an enzyme is mixed with a large excess of the substrate, the enzyme-substrate intermediate builds up in a fast initial transient. Then the reaction achieves a steady-state kinetics in which enzyme substrate intermediates remains approximately constant over time and the reaction rate changes relatively slowly. Rates are measured for a short period after the attainment of the quasi-steady state, typically by monitoring the accumulation of product with time. Because the measurements are carried out for a very short period and because of the large excess of substrate, the approximation free substrate is approximately equal to the initial substrate can be made. The initial rate experiment is the simplest to perform and analyze, being relatively free from complications such as back-reaction and enzyme degradation. It is therefore by far the most commonly used type of experiment in enzyme kinetics.

(2) Progress curve experiments. In these experiments, the kinetic parameters are determined from expressions for the species concentrations as a function of time. The concentration of the substrate or product is recorded in time after the initial fast transient and for a sufficiently long period to allow the reaction to approach equilibrium. We note in passing that, while they are less common now, progress curve experiments were widely used in the early period of enzyme kinetics.

(3) Transient kinetics experiments. In these experiments, reaction behaviour is tracked during the initial fast transient as the intermediate reaches the steady-state kinetics period. These experiments are more difficult to perform than either of the above two classes because they require rapid mixing and observation techniques.

(4) Relaxation experiments. In these experiments, an equilibrium mixture of enzyme, substrate and product is perturbed, for instance by a temperature, pressure or pH jump, and the return to equilibrium is monitored. The analysis of these experiments requires consideration of the fully reversible reaction. Moreover, relaxation experiments are relatively insensitive to mechanistic details and are thus not typically used for mechanism identification, although they can be under appropriate conditions.

Enzyme assays can be split into two groups according to their sampling method: continuous assays, where the assay gives a continuous reading of activity, and discontinuous assays, where samples are taken, the reaction stopped and then the concentration of substrates/products determined.

Continuous assays

Continuous assays are most convenient, with one assay giving the rate of reaction with no further work necessary. There are many different types of continuous assays.

Spectrophotometric

In spectrophotometric assays, you follow the course of the reaction by measuring a change in how much light the assay solution absorbs. If this light is in the visible region you can actually see a change in the color of the assay, these are called colorimetric assays. The MTT assay, a redox assay using a tetrazolium dye as substrate is an example of a colorimetric assay. Ultraviolet-visible spectroscopy or ultraviolet-visible spectrophotometry (UV/ VIS) involves the spectroscopy of photons and spectrophotometry. ... MTT assay is a laboratory test and a standard colorimetric assay (an assay which measures changes in colour) for measuring cellular proliferation (cell growth). ...

UV light is often used, since the common coenzymes NADH and NADPH absorb UV light in their reduced forms, but do not in their oxidised forms. An oxidoreductase using NADH as a substrate could therefore be assayed by following the decrease in UV absorbance at 340 nm as it consumes the coenzyme.^[2] Space-filling model of NADH Nicotinamide adenine dinucleotide (NAD+) is an important coenzymes found in cells. ... Space-filling model of NADH Nicotinamide adenine dinucleotide (NAD+) is an important coenzymes found in cells. ... Illustration of a redox reaction Redox (shorthand for oxidation/reduction reaction) describes all chemical reactions in which atoms have their oxidation number (oxidation state) changed. ... Illustration of a redox reaction Redox (shorthand for oxidation/reduction reaction) describes all chemical reactions in which atoms have their oxidation number (oxidation state) changed. ... In biochemistry, an oxidoreductase is an enzyme that catalyzes the transfer of electrons from one molecule (the oxidant, also called the hydrogen donor or electron donor) to another (the reductant, also called the hydrogen acceptor or electron acceptor). ...

Direct versus coupled assays

Figure 1: Coupled assay for hexokinase using glucose-6-phosphate dehydrogenase.

Even when the enzyme reaction does not result in a change in the absorbance of light, it can still be possible to use a spectrophotometric assay for the enzyme by using a coupled assay. Here, the product of one reaction is used as the substrate of another, easily-detectable reaction. For example, figure 1 shows the coupled assay for the enzyme hexokinase, which can be assayed by coupling its production of glucose-6-phosphate to NADPH production, using glucose-6-phosphate dehydrogenase. Image File history File links Coupled_assay. ... Image File history File links Coupled_assay. ... A hexokinase is an enzyme that phosphorylates a six-carbon sugar, a hexose, to a hexose phosphate. ... Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked recessive hereditary disease featuring nonimmune hemolytic anemia in response to a number of causes. ...

Fluorimetric

Fluorescence is when a molecule emits light of one wavelength after absorbing light of a different wavelength. Fluorometric assays use a difference in the fluorescence of substrate from product to measure the enzyme reaction. These assays are in general much more sensitive than spectrophotometric assays, but can suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed to light. Fluorescence induced by exposure to ultraviolet light in vials containing various sized Cadmium selenide (CdSe) quantum dots. ... The wavelength is the distance between repeating units of a wave pattern. ... Fluorescence induced by exposure to ultraviolet light in vials containing various sized Cadmium selenide (CdSe) quantum dots. ...

An example of these assays is again the use of the nucleotide coenzymes NADH and NADPH. Here, the reduced forms are fluorescent and the oxidised forms non-fluorescent. Oxidation reactions can therefore be followed by a decrease in fluorescence and reduction reactions by an increase.^[3] Synthetic substrates that release a fluorescent dye in an enzyme-catalyzed reaction are also available, such as 4-methylumbelliferyl-β-D-glucuronide for assaying β-galactosidase. A β-galactosidase is a type of hydrolase that that catalyzes the cleavage of terminal, β-linked, nonreducing galactose residues from a variety of substrates, including ganglioside GM1, lactosylceramides, lactose, and various glycoproteins and oligosaccharides. ...

Calorimetric

Chemiluminescence of Luminol

Calorimetry is the measurement of the heat released or absorbed by chemical reactions. These assays are very general, since many reactions involve some change in heat and with use of a microcalorimeter, not much enzyme or substrate is required. These assays can be used to measure reactions that are impossible to assay in any other way.^[4] Image File history File links Size of this preview: 456 × 600 pixelsFull resolution (960 × 1263 pixel, file size: 157 KB, MIME type: image/jpeg) File history Legend: (cur) = this is the current file, (del) = delete this old version, (rev) = revert to this old version. ... Image File history File links Size of this preview: 456 × 600 pixelsFull resolution (960 × 1263 pixel, file size: 157 KB, MIME type: image/jpeg) File history Legend: (cur) = this is the current file, (del) = delete this old version, (rev) = revert to this old version. ... The world’s first ice-calorimeter, used in the winter of 1782-83, by Antoine Lavoisier and Pierre-Simon Laplace, to determine the heat evolved in various chemical changes; calculations which were based on Joseph Black’s prior discovery of latent heat. ...

Chemiluminescent

Chemiluminescence is the emission of light by a chemical reaction. Some enzyme reactions produce light and this can be measured to detect product formation. These types of assay can be extremely sensitive, since the light produced can be captured by photographic film over days or weeks, but can be hard to quantify, because not all the light released by a reaction will be detected. A chemoluminescent reaction carried out in an erlenmeyer flask producing a large amount of light. ...

The detection of horseradish peroxidase by enzymatic chemiluminescence (ECL) is a common method of detecting antibodies in western blotting. Another example is the enzyme luciferase, this is found in fireflies and naturally produces light from its substrate luciferin. Glutathione Peroxidase 1 A peroxidase (eg. ... Picture of a Western blot with 5 vertical lanes A Western blot is a method in molecular biology/biochemistry to detect protein in a given sample of tissue homogenate or extract. ... Luciferase is a generic name for enzymes commonly used in nature for bioluminescence. ...

Discontinuous assays

Discontinuous assays are when samples are taken from an enzyme reaction at intervals and the amount of product production or substrate consumption is measured in these samples.

Radiometric

Radiometric assays measure the incorporation of radioactivity into substrates or its release from substrates. The radioactive isotopes most frequently used in these assays are ¹⁴C, ³²P, ³⁵S and ¹²⁵I. Since radioactive isotopes can allow the specific labelling of a single atom of a substrate, these assays are both extremely sensitive and specific. They are frequently used in biochemistry and are often the only way of measuring a specific reaction in crude extracts (the complex mixtures of enzymes produced when you lyse cells). Radioactivity may mean: Look up radioactivity in Wiktionary, the free dictionary. ... A radionuclide is an atom with an unstable nucleus. ...

Radioactivity is usually measured in these procedures using a scintillation counter. A scintillation counter measures ionizing radiation. ...

Chromatographic

Chromatographic assays measure product formation by separating the reaction mixture into its components by chromatography. This is usually done by high-performance liquid chromatography (HPLC). Although this approach can need a lot of material, its sensitivity can be increased by labelling the substrates/products with a radioactive or fluorescent tag. Pictured is a sophisticated gas chromatography system. ... A HPLC. From left to right: A pumping device generating a gradient of two different solvents, a steel enforced column and an apparatus for measuring the absorbance. ...

Factors to control in assays

• Salt Concentration: Most enzymes can not tolerate extremely high salt concentrations. The ions interfere with the weak ionic bonds of proteins. As usual there are exceptions such as the halophilic (salt loving) algae and bacteria.

• Effects of Temperature: All enzymes work within a range of temperature specific to the organism. Increases in temperature generally lead to increases in reaction rates. There is a limit to the increase because higher temperatures lead to a sharp decrease in reaction rates. This is due to the denaturating (alteration) of protein structure resulting from the breakdown of the weak ionic and hydrogen bonding that stabilize the three dimensional structure of the enzyme. The optimum temperature for human enzymes is between 35 and 40 °C. The average temperature for humans is 37 °C. Human enzymes start to denature quickly at temperatures above 40 °C. Bacteria found in the hot springs of Yellowstone National Park have optimum temperatures of 70 °C. Temperature loving bacteria are called thermophilic. Denaturating of enzymes, regardless of the cause, leads to the death of the cell.

• Effects of pH: Most enzymes are sensitive to pH and have specific ranges of activity. All have an optimum pH. The pH can stop enzyme activity by denaturating (altering) the three dimensional shape of the enzyme by breaking weak bonds such as ionic, and hydrogen. Most enzymes function between a pH of 6 and 8; however pepsin in the stomach works best at a pH of 2 and trypsin at a pH of 8. Bacteria that thrive at lower pH values are said to be acidophilic (acid loving).

• Enzyme Saturation: Increasing the substrate concentration increases the rate of reaction (enzyme activity). However, enzyme saturation limits reaction rates. An enzyme is saturated when the active sites of all the molecules are occupied most of the time. At the saturation point, the reaction will not speed up, no matter how much additional substrate is added. The graph of the reaction rate will plateau. It can be increased if more enzyme is added.] concentration can inactivate an enzyme. Typical enzymes are active in salt concentrations of 1-500 mM.

Electron configurations of lithium and fluorine. ... A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... A seaweed (Laurencia) up close: the branches are multicellular and only about 1 mm thick. ... Phyla Actinobacteria Aquificae Chlamydiae Bacteroidetes/Chlorobi Chloroflexi Chrysiogenetes Cyanobacteria Deferribacteres Deinococcus-Thermus Dictyoglomi Fibrobacteres/Acidobacteria Firmicutes Fusobacteria Gemmatimonadetes Lentisphaerae Nitrospirae Planctomycetes Proteobacteria Spirochaetes Thermodesulfobacteria Thermomicrobia Thermotogae Verrucomicrobia Bacteria (singular: bacterium) are unicellular microorganisms. ... A representation of the 3D structure of myoglobin, showing coloured alpha helices. ... From ancient Greece (Ionic) An Ionian is a member of one of the four great divisions of the ancient Greek people. ... In chemistry, a hydrogen bond is a type of attractive intermolecular force that exists between two partial electric charges of opposite polarity. ... Yellowstone National Park is the centerpiece of the Greater Yellowstone Ecosystem, the largest intact ecosystem in the Earths northern temperate zone. ... Thermophiles produce some of the bright colors of Grand Prismatic Spring, Yellowstone National Park A thermophile is an organism – a type of extremophile – which thrives at relatively high temperatures, up to about 60 °C. Many thermophiles are archaea. ... The correct title of this article is . ... From ancient Greece (Ionic) An Ionian is a member of one of the four great divisions of the ancient Greek people. ... In biochemistry, a substrate is a molecule upon which an enzyme acts. ...

List of enzyme assays

• MTT assay
• Overlay assay

MTT assay is a laboratory test and a standard colorimetric assay (an assay which measures changes in colour) for measuring cellular proliferation (cell growth). ...

See also

• Restriction enzyme
• DNase footprinting assay
• Enzyme kinetics

A restriction enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA. The enzyme makes two incisions, one through each of the sugar-phosphate backbones (i. ... A DNase footprinting assay [1]is a technique from molecular biology that detects DNA-protein interaction using the fact that a protein bound to DNA will often protect that DNA from enzymatic cleavage. ... Dihydrofolate reductase from with its two substrates, dihydrofolate (right) and NADPH (left), bound in the active site. ...

References

1. ^ Schnell, S., Chappell, M. J., Evans, N. D. and Roussel, M. R. The mechanism distinguishability problem in biochemical kinetics: The single-enzyme, single-substrate reaction as a case study. Comptes Rendus Biologies 2006; 329, 51-61. DOI: 10.1016/j.crvi.2005.09.005
2. ^ Bergmeyer, H. U. "Methods of Enzymatic Analysis", Vol. 4, Academic Press (New York, NY:1974), pp.2066-2072.
3. ^ Passonneau, J. V., and Lowry, O. H. "Enzymatic Analysis. A Practical Guide", Humana Press (Totowa, NJ:1993), pp.85-110.
4. ^ Todd MJ, Gomez J. Enzyme kinetics determined using calorimetry: a general assay for enzyme activity? Anal Biochem. 2001 Sep 15;296(2):179-87.